Structure of b-1,4-galactosyltransferase (Gal-T1) and Lactose Synthase (LS) and Design of Novel Glycosyltransferases: Defective glycan synthesis has been shown to have serious pathological consequences and result in several human diseases. Our aim has been to determine the structure of galactosyltransferase family members which are involved in the synthesis of complex oligosaccharide structures of glycoconjugates and correlate their structure with function. Gal-T1, a member of galactosyltransferase sub-family, transfers galactose (Gal) from UDP-Gal to an acceptor GlcNAc in the presence of Mn2+ion. a-Lactalbumin (LA), a mammary gland-specific protein, interacts with Gal-T1 enzyme and forms a lactose synthase (LS) complex that alters the acceptor specificity of the enzyme towards glucose (Glc) to produce lactose. Structural and biochemical investigations from our laboratory on Gal-T1 and LS have revealed that they are akin to an exquisite mechanical device with two well-coordinated flexible loops that are contained within the Gal-T1 catalytic domain. The smaller one has a Trp residue (Trp314) flanked by glycine residues. The larger one comprises amino acid residues 345 to 365. Upon substrate binding, the Trp314 side chain moves to lock the sugar nucleotide in the binding site, while the large loop undergoes a conformational change, masking the sugar nucleotide binding site, and creates: 1) the oligosaccharide binding cavity; 2) a protein-protein interacting site for the enzyme's partner, LA; and 3) a metal ion binding site. During conformational change, His347 is positioned in such a way that it can partake in coordinating the Mn2+ion with Met344 and Asp254 of D252VD254 sequence. Site-directed mutagenesis and kinetic analysis, and the crystal structures of the mutants show that Asp254 and His347 strongly bind metal ligand, while Met344 coordinates less strongly and can be substituted by serine, glutamine or alanine. The Asp252 of D252VD254 sequence is not involved in Mn2+ion binding; instead it binds to the galactose moiety of UDP-Gal. Only in conformation II do Gal-T1 and LA form the LS complex, enabling Gal-T1 to choose the new substrate glucose. LA holds and puts Glc right in the acceptor binding site of Gal-T1, which then maximizes the interactions with Glc, thereby making it a preferred acceptor for the LS reaction. The interaction of LA with Gal-T1 in conformation II also stabilizes the sugar-nucleotide-enzyme complex, kinetically enhancing the sugar transfer, even from the less preferred sugar nucleotides. Gal-T family members can behave as specific lectins: The conformational change that masks the sugar nucleotide binding site can also be induced by the acceptor alone. The conformational change in the large loop puts a lid - a cover - on the sugar nucleotide binding site, while simultaneously creating an extended sugar binding site that can accommodate an N-glycan, penta-saccharide in length, thus enabling the protein to act as a specific lectin. Each member of the Gal-T family shows sequence variation in its oligosaccharide binding site. Each is expressed in a tissue-specific manner and shows preferences for specific oligosaccharides. Given these properties they can act as tissue-specific lectins. The molecules like LA may interact with the LA binding site, which is also the oligosaccharide binding site, and may act as specific lectin inhibitors by competing for the binding of the oligosaccharide. Structure based design of novel glycosyltransferases: The information derived from the crystal structures of Gal-T1 with less preferred donor or acceptor substrates has been used to design the mutants of Gal-T1 that have preferences for different donor and acceptor substrates and has allowed us to synthesize oligosaccharides that are otherwise chemically difficult to synthesize. The crystal structure of the Gal-T1 LA complex with UDP-GalNAc was determined at 2.1 ? resolution. The structure reveals that the UDP-GalNAc binding to Gal-T1 is quite similar to the binding of UDP-Gal to Gal-T1, except for an additional hydrogen bond that is formed between the N-acetyl group of GalNAc moiety with the side chain hydroxyl group of Tyr289. It is reasoned that this additional hydrogen bond would account for the low GalNAc transferase activity of Gal-T1. This condition was substantiated by the studies on the mutants of Tyr289. The mutant Y289L exhibits enhanced GalNAc-transferase activity that approaches 100% of the wild-type Gal-T activity even while retaining its 100% Gal-T activity. The steady state kinetic analyses on the Leu289 mutant indicate that in the Gal-T reaction, the Km for GlcNAc has increased nearly twentyfold compared to wild-type, whereas the donor catalytic efficiency (kcat/KA) is quite similar for both donors, UDP-Gal and UDP-GalNAc. This study demonstrates that in the Gal-T family the Tyr289/Phe289 residue largely determines the sugar donor specificity. In a cell, a single point mutation of this residue has the potential to change the sugar donor specificity of Gal-T1, which may have serious implications in cellular processes.